1. Technical Field
The present invention relates to biotechnology, specifically to a method for producing L-amino acids by fermentation using glycerol, and more specifically to a method for producing L-amino acids using bacteria having enhanced ability to utilize glycerol. An inexpensive carbon source including glycerol could be utilized for commercial production of L-amino acids.
2. Background Art
Conventionally, L-amino acids have been industrially produced by a process of fermentation using strains of different microorganisms. The fermentation media for the process should contain sufficient amounts of different sources of carbon and nitrogen.
Traditionally various carbohydrates such as hexoses, pentoses, trioses; various organic acids and alcohols are used as a carbon source. Hexoses include glucose, fructose, mannose, sorbose, galactose and the like. Pentoses include arabinose, xylose, ribose and the like. But abovementioned carbohydrates and other traditional carbon sources, such as molasses, corn, sugarcane, starch, its hydrolysate, etc., used in the industry are still relatively expensive and a reduction in price of L-amino acid produced is desired.
Glycerol, especially glycerol obtained as by-product of biodiesel production, is a favorable feedstock for L-amino acid production because it is both readily available and less expensive than carbohydrates, corn, sugarcane or other sources of carbon. It is known also that bacteria can use glycerol as a carbon source for growth. (Ito T. et al, J Biosci Bioeng., 100, 3, 260-5 (2005)).
Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. Glycerol kinase (EC2.7.1.30) encoded by the glpK gene is a component of regulatory network in E. coli by which glucose and other carbon sources control the utilization of glycerol and the gene expression that is needed for glycerol metabolism. (Escherichia coli and Salmonella. 2nd edition ASM Press Washington, D.C.). The proteins involved in glycerol metabolism are encoded by the glp regulon, which contains five operons located at three different chromosomal loci. Glucose modulation of glycerol utilization involves both regulation of transcription and posttranslational control of glycerol kinase catalytic activity. Transcription of the regulon is negatively controlled by a specific repressor encoded by the glpR gene.
It is known that cytoplasmic glycerol is immediately phosphorylated by the ATP-dependent glycerol kinase, which is present in its enzymatically active form associated with the glycerol facilitator GlpF (Voegele, R. T. et al, J. Bacteriol 175, 4, 1087-1094 (1993))). Furthermore, the glycerol kinase is subject to feedback inhibition by fructose-1,6-bisphosphate (fructose-1,6 diphosphate, FBP). Accordingly, the activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. 
The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. It was found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation is different, depending on the presence or absence of the facilitator protein. It was concluded that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria (Voegele, R. T. et al, J. Bacteriol., 175, 4, 1087-1094 (1993)).
A mutant strain which produces a glycerol kinase resistant to inhibition by fructose-1,6-bisphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon. Pittigrew et al. identified the Escherichia coli glycerol kinase mutation G304S which lost sensitivity to inhibition by FBP (Pettigrew, D. W., Liu, W. Z., Holmes, C., Meadow, N. D., and Roseman, S., J. Bacteriol. 178, 10, 2846-52 (1996)). Honisch et. al. identified the mutation G231D in glycerol kinase of an adaptively evolved strain, and kinetically characterized wild type glycerol kinase and G231D mutant. Kinetic studies for G231D variant show a 12-fold increase in glycerol kinase activity and simultaneous increase in tolerance toward the allosteric inhibitor fructose-1,6-bisphosphate (Honisch, C. et. al., Genome Research, 14: 2495-2502 (2004)).
Furthermore, adaptation of E. coli to glycerol media was tested, and some mutations which allowed for growth in the glycerol medium were determined (Herring C. D. et al, Nat. Genet., 38 (12): 1406-1412 (2006). Epub 2006 Nov. 5).
It is known also that attenuation of glpR is effective for producing L-amino acid production by fermentation in a glycerol containing medium. (EP1715056A1)
However, such known mutants are not sufficient for L-amino acid production from glycerol.
Furthermore, at present, there are no reports describing bacteria having a synergetic effect by combination of a mutation in glycerol kinase and attenuation of glpR on L-amino acid production from glycerol.